Abstract

Myotonic dystrophy type 1 (DM1) is a complex neuromuscular disease characterized by skeletal muscle wasting, weakness, and myotonia. DM1 is caused by the accumulation of CUG repeats, which alter the biological activities of RNA-binding proteins, including CUG-binding protein 1 (CUGBP1). CUGBP1 is an important skeletal muscle translational regulator that is activated by cyclin D3–dependent kinase 4 (CDK4). Here we show that mutant CUG repeats suppress Cdk4 signaling by increasing the stability and activity of glycogen synthase kinase 3β (GSK3β). Using a mouse model of DM1 (HSALR), we found that CUG repeats in the 3′ untranslated region (UTR) of human skeletal actin increase active GSK3β in skeletal muscle of mice, prior to the development of skeletal muscle weakness. Inhibition of GSK3β in both DM1 cell culture and mouse models corrected cyclin D3 levels and reduced muscle weakness and myotonia in DM1 mice. Our data predict that compounds normalizing GSK3β activity might be beneficial for improvement of muscle function in patients with DM1.

Figure 1

(A) Western blot analyses of the total protein extracts from patients with normal muscle histopathology (N1, N2) and from patients affected with DM1 (D1, D2) were performed, with antibodies shown on the right. (B) Examination of GSK3β/cyclin D3/CUGBP1 pathway in skeletal muscles of 6 additional patients with DM1. Western blotting was performed with antibodies to cyclin D3 (Cyc D3), GSK3β, p-S9–GSK3β, CUGBP1, and actin. (C) Average levels of cyclin D3, GSK3β, p-S9–GSK3β, and CUGBP1 presented as ratios to actin. The standard deviations represent quantitation of protein expression based on 3 experiments. (D) Phosphorylation of cyclin D3 at T283 is increased in DM1 skeletal muscle biopsies. IP–Western blot assay. Cyclin D3 was precipitated with antibodies to total cyclin D3. Cyclin D3 IPs were divided into two portions and examined by Western blot assay with antibodies to Rb, p-T286 (recognizing p-T283 in cyclin D3), and total cyclin D3. The signal of IgGs is the control for antibodies used for immunoprecipitation. Since cyclin D3 is reduced in DM1, the amount of protein from DM1 muscle tissue used for IP was 6-fold higher than that isolated from normal muscle. Ratios of p-T283–cyclin D3 (E) and Rb (F) to total cyclin D3 were determined by quantitating protein expression from D. The standard deviations show values based on 3 experiments.